Compositions comprising a p75 tumor necrosis factor receptor/ig fusion protein

ABSTRACT

The present invention relates to an aqueous pharmaceutical composition suitable for long-term storage of polypeptides containing an Fc domain of an immunoglobulin, methods of manufacture, methods of administration and kits containing same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/341,962, filedNov. 2, 2016, which is continuation of U.S. Ser. No. 14/478,926, filedSep. 5, 2014, now U.S. Pat. No. 9,518,111, issued Dec. 13, 2016, whichis a divisional of U.S. Ser. No. 13/401,496, filed Feb. 21, 2012, nowU.S. Pat. No. 8,828,947, issued Sep. 9, 2014, which is a continuation ofU.S. Ser. No. 12/632,690, filed Dec. 7, 2009, now issued as U.S. Pat.No. 8,119,604, issued Feb. 21, 2012, which is a divisional ofapplication U.S. Ser. No. 11/784,538, filed Apr. 6, 2007, now issued asU.S. Pat. No. 7,648,702, issued Jan. 19, 2010 which is a continuation ofapplication U.S. Ser. No. 10/376,576, filed Feb. 27, 2003, nowabandoned, which claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Application No. 60/360,257, filed Feb. 27, 2002, each ofwhich is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to an aqueous pharmaceutical compositionsuitable for long-term storage of polypeptides containing an Fc domainof an immunoglobulin, methods of manufacture of the composition, methodsof administration and kits containing same.

BACKGROUND

After production, polypeptides must typically be stored prior to theiruse. Frequently, when stored for extended periods polypeptides areunstable in solution (Manning et al., 1989, Pharm. Res. 6:903-918).Accordingly, additional processing steps have been developed to allowfor a longer shelf life including drying, e.g., lyophilization. However,lyophilized pharmaceutical compositions are less convenient for the enduser.

Typical practices to improve polypeptide stability can be addressed byvarying the concentration of elements with the formulation, or by addingexcipients to modify the formulation (U.S. Pat. Nos. 5,580,856 and6,171,586). The use of additives, while improving storage, can stillresults in inactive polypeptides. In addition, in the case oflyophilization, the rehydration step can introduce conditions thatresult in inactivation of the polypeptide by, for example, aggregationor denaturation (Hora et al., 1992, Pharm. Res., 9:33-36; Liu et al.,1991, Biotechnol. Bioeng., 37:177-184). In fact, aggregation ofpolypeptides is undesirable as it may result in immunogenicity (Clelandet al., 1993, Crit. Rev. Therapeutic Drug Carrier Systems, 10:307-377;and Robbins et al., 1987, Diabetes, 36:838-845).

The present invention addresses these issues by providing a novel stableliquid formulation that allows long term storage of a polypeptidecontaining an Fc domain of an immunoglobulin.

SUMMARY

The invention relates, in part, to a stable aqueous pharmaceuticalcomposition comprising a therapeutically effective amount of an Fcdomain containing polypeptide, an aggregation inhibitor selected fromthe group consisting of L-arginine and L-cysteine. Optionally, thecomposition can include a buffer, a tonicity modifier and one or moreexcipients. In one aspect, the buffer maintains the composition pH at arange of about 6.0 and about 7.0. Preferably, the Fc domain containingpolypeptide is stable in the present formulation for at least threemonths at 2-8° C. and/or is stable following one or more freezing andthawing cycles of the formulation.

The invention also relates to a method of formulating a pharmaceuticalcomposition, the composition an Fc domain containing polypeptide with anaggregation inhibitor selected from the group consisting of L-arginineand L-cysteine. Optionally, one can also add to the pharmaceuticalcomposition a buffer, a tonicity modifier and/or an excipient. In oneaspect, the pharmaceutical composition is formulated at a pH rangebetween pH 6.0 and 7.0.

The invention also relates to a method of treating a mammal comprisingadministering a therapeutically effective amount of the pharmaceuticalcomposition described herein, wherein the mammal has a disease ordisorder that can be beneficially treated with a Fc domain containingpolypeptide in the composition.

The invention also relates to a method of accelerated stability testingof an Fc domain containing polypeptide in a pharmaceutical compositionof the invention comprising the steps of storing the composition at 37°C. for one month and measuring the stability of the polypeptide.

In another embodiment, the present invention is directed to a kit orcontainer, which contains an aqueous pharmaceutical composition of theinvention. The kit can also be accompanied by instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Size exclusion chromatography (SEC) data for lots A, B, C and D,and lot 1 stored for up to 1 year at 2-8° C.

FIG. 2: SEC data for lots A, B, C and D, and lot 1 stored for up to 1year at 37° C.

FIG. 3: Denatured SEC (dSEC) data for lots A, B, C and D, and lot 1stored for up to 1 year at 2-8° C.

FIG. 4: dSEC data for lots A, B, C and D, and lot 1 stored for up to 1year at 37° C.

FIG. 5: Hydrophobic interaction chromatography (HIC) Peak 1 and Pre-peak1 data for lots A, B, C and D, and lot 1 stored for up to 1 year at 2-8°C.

FIG. 6: HIC Peak 1 and Pre-peak 1 data for lots A, B, C and D, and lot 1stored for up to 1 year at 37° C.

FIG. 7: HIC Peak 2 data for lots A, B, C and D, and lot 1 stored for upto 1 year at 2-8° C.

FIG. 8: HIC Peak 2 data for lots A, B, C and D, and lot 1 stored for upto 1 year at 37° C.

FIG. 9: HIC Peak 3 data for lots A, B, C and D, and lot 1 stored for upto 1 year at 2-8° C.

FIG. 10: HIC Peak 3 data for lots A, B, C and D, and lot 1 stored for upto 1 year at 37° C.

FIG. 11: Binding activity of lots A, B, C and D, and lot 1 stored for 12months at −70, 2-8, 30, and 37° C.

FIG. 12: Bioactivity of lots A, B, C and D, and lot 1 stored for 12months at −70, 2-8, 30, and 37° C.

DETAILED DESCRIPTION

In long-term storage of pharmaceutical compositions containingpolypeptides, including aqueous and lyophilized formulations, activepolypeptides can be lost due to aggregation and/or degradation. Thus,the present invention is directed to an aqueous formulation thatsurprisingly allows for stable long-term storage of a pharmaceuticalcomposition wherein the active ingredient in the composition is apolypeptide having an Fc domain of an antibody. This formulation isuseful, in part, because it is more convenient to use for the patient,as this formulation does not require any extra steps such asrehydrating.

As used herein, the phrase “pharmaceutical composition” is understood torefer to a formulation comprised of a polypeptide prepared such that itis suitable for injection and/or administration into a patient in needthereof. More particularly, a pharmaceutical composition issubstantially sterile and does not contain any agents that are undulytoxic or infectious to the recipient. Further, it is to be understoodthat, as used herein, a solution or liquid formulation is meant to meana liquid preparation that contains one or more chemical substancesdissolved in a suitable solvent or mixture of mutually misciblesolvents.

In addition, as used herein, the term “about” is understood to mean thatthere can be variation in the concentration of a component of thedescribed formulation that can be to 5%, 10%, 15% or up to and including20% of the given value. For example, if a formulation has about 10 mg/mlof an Fc domain containing polypeptide, this is understood to mean thata formulation can have between 8 to 12 mg/ml of the stated polypeptide.

In one embodiment, the formulation is comprised of an Fc domaincontaining polypeptide, an aggregation inhibitor selected from groupconsisting of L-arginine and L-cysteine, and, optionally, a buffer, atonicity modifier and additional excipients as necessary. L-arginine hasbeen used to assist refolding of insoluble polypeptides, particularlythose expressed to high levels in inclusion bodies in bacteria. However,L-arginine has not been utilized successfully to enhance stability of Fcdomain containing polypeptides in pharmaceutical compositions (Soejimaet al., 2001, J. Biochem., 130:369-277).

It is contemplated that the preparation of the composition should bedone in consideration of limiting injection site discomfort. It isfurther contemplated that additional active ingredients can also beincluded in the presently described composition, for example, to reduceinjection site discomfort. Such active ingredients include, but are notlimited to non-steroidal anti-inflammatory drugs such as, for example,tromethamine, in an appropriate dosage.

Polypeptides

In a particular embodiment the Fc domain containing polypeptide is asoluble form of the TNF receptor fused to an Fc domain (TNFR:Fc),however, it is to be understood that any polypeptide containing an Fcdomain is suitable for use in the instant formulation. A commerciallyavailable TNFR:Fc is known as etanercept (Enbrel®, Immunex Corporation),which is a dimeric fusion polypeptide consisting of the extracellularligand-binding portion of the human 75 kilodalton (p′75) tumor necrosisfactor receptor (TNFR) linked to the Fc portion of human IgG1. The Fccomponent of etanercept contains the constant heavy 2 (CH2) domain, theconstant heavy 3 (CH3) domain and hinge region, but not the constantheavy 1 (CH1) domain of human IgG1. It is to be understood that an Fcdomain can contain one or all of the domains described above. Etanerceptis produced by recombinant DNA technology in a Chinese hamster ovary(CHO) mammalian cell expression system. It consists of 934 amino acidsand has an apparent molecular weight of approximately 150 kilodaltons(Physicians Desk Reference, 2002, Medical Economics Company Inc.).

Other polypeptides specifically contemplated for formulation accordingto the invention include recombinant fusion polypeptides comprising atleast a portion of an Fc domain of an antibody. A polypeptide fused toan Fc domain and identical to or substantially similar to one of thefollowing polypeptides is suitable for use in the present pharmaceuticalcomposition: a flt3 ligand, a CD40 ligand, erythropoeitin,thrombopoeitin, calcitonin, Fas ligand, ligand for receptor activator ofNF-kappa B (RANKL), tumor necrosis factor (TNF)-relatedapoptosis-inducing ligand (TRAIL), thymic stroma-derived lymphopoietin,granulocyte colony stimulating factor, granulocyte-macrophage colonystimulating factor, mast cell growth factor, stem cell growth factor,epidermal growth factor, RANTES, growth hormone, insulin,insulinotropin, insulin-like growth factors, parathyroid hormone,interferons, nerve growth factors, glucagon, interleukins 1 through 18,colony stimulating factors, lymphotoxin-β, tumor necrosis factor (TNF),leukemia inhibitory factor, oncostatin-M, and various ligands for cellsurface molecules ELK and Hek (such as the ligands for eph-relatedkinases or LERKS).

Polypeptides suitable for formulation according to the invention alsoinclude recombinant fusion polypeptides comprising an Fc domain of anantibody plus a receptor for any of the above-mentioned polypeptides orpolypeptides substantially similar to such receptors. These receptorsinclude: both forms of TNFR (referred to as p55 and p75), Interleukin-1receptors (type 1 and 2), Interleukin-4 receptor, Interleukin-15receptor, Interleukin-17 receptor, Interleukin-18 receptor,granulocyte-macrophage colony stimulating factor receptor, granulocytecolony stimulating factor receptor, receptors for oncostatin-M andleukemia inhibitory factor, receptor activator of NF-kappa B (RANK),receptors for TRAIL (TRAIL receptors 1, 2, 3, and 4), and receptors thatcomprise death domains, such as Fas or Apoptosis-Inducing Receptor(AIR).

Other polypeptides suitable for use in the present formulation includedifferentiation antigens (referred to as CD polypeptides) or theirligands or polypeptides substantially similar to either of these, whichare fused to an Fc domain of an antibody. Such antigens are disclosed inLeukocyte Typing VI (Proceedings of the VIth International Workshop andConference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 1996).Similar CD polypeptides are disclosed in subsequent workshops. Examplesof such antigens include CD27, CD30, CD39, CD40, and ligands thereto(CD27 ligand, CD30 ligand, etc.). Several of the CD antigens are membersof the TNF receptor family, which also includes 41BB ligand and OX40.The ligands are often members of the TNF family, as are 41BB ligand andOX40 ligand. Accordingly, members of the TNF and TNFR families can beformulated according to the present invention.

Enzymatically active polypeptides or their ligands can also beformulated according to the invention. Examples include recombinantfusion polypeptides comprising an Fc domain of an antibody fused to allor part of one of the following polypeptides or their ligands or apolypeptide substantially similar to one of these:metalloproteinase-disintegrin family members, various kinases,glucocerebrosidase, superoxide dismutase, tissue plasminogen activator,Factor VIII, Factor IX, apolipoprotein E, apolipoprotein A-I, globins,an IL-2 antagonist, alpha-1 antitrypsin, TNF-alpha Converting Enzyme,ligands for any of the above-mentioned enzymes, and numerous otherenzymes and their ligands.

The formulations and methods of the invention can also be used toprepare pharmaceutical compositions comprising antibodies, humanantibodies, humanized antibodies, chimeric antibodies, i.e. antibodieshaving human constant antibody immunoglobulin domains coupled to one ormore murine variable antibody immunoglobulin domain, and/or non-humanantibodies, or fragments thereof. Specific examples of antibodiessuitable for use in the present formulation include commerciallyavailable antibodies such as muromonab-CD3 (Orthoclone OKT-3®, OrthoBiotech), abciximab (ReoPro®, Lilly), rituximab (Rituxan®, IDEC),dacliximab (Zenapax®, Roche Laboratories), basiliximab (Simulect®,Novartis), infliximab (Remicade®, Centocor), palivizumab (Synagis®,MedImmune), trastuzumab (Herceptin®, Genentech), gemtuzuman ozogamicin(Mylotarg™, Wyeth-Ayerst), and alemtuzumab (Campath®, Berlex). Currentlyeach of the foregoing is available either as a lyophilized powderrequiring rehydration or as a concentrate requiring dilution prior toadministration. The present formulation obviates the need for anymanipulations prior to administration, e.g., rehydrating or dilution,while preserving stability of the active ingredients over long-termstorage.

The pharmaceutical composition of the invention can also be used tostore polypeptides comprising an antibody conjugated to a cytotoxic orluminescent substance. Such substances include: maytansine derivatives(such as DM1); enterotoxins (such as a Staphylococcal enterotoxins);iodine isotopes (such as iodine-125); technetium isotopes (such asTc-99m); cyanine fluorochromes (such as Cy5.5.18); andribosome-inactivating polypeptides (such as bouganin, gelonin, orsaporin-56).

Examples of antibodies or antibody/cytotoxin or antibody/luminophoreconjugates contemplated for use in the invention include those thatrecognize one or more of the following antigens: CD2, CD3, CD4, CD8,CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80(B7.1), CD86 (B7.2), CD147, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4receptor, IL-6 receptor, IL-13 receptor, PDGF-β, VEGF, TGF, TGF-β2,TGF-β1, EGF receptor, VEGF receptor, C5 complement, IgE, tumor antigenCA125, tumor antigen MUC1, PEM antigen, LCG (which is a gene productthat is expressed in association with lung cancer), HER-2, atumor-associated glycoprotein TAG-72, the SK-1 antigen, tumor-associatedepitopes that are present in elevated levels in the sera of patientswith colon and/or pancreatic cancer, cancer-associated epitopes orpolypeptides expressed on breast, colon, squamous cell, prostate,pancreatic, lung, and/or kidney cancer cells and/or on melanoma, glioma,or neuroblastoma cells, TRAIL receptors 1, 2, 3 and 4, the necrotic coreof a tumor, integrin alpha 4 beta 7, the integrin VLA-4, B2 integrins,TNF-α, the adhesion molecule VAP-1, epithelial cell adhesion molecule(EpCAM), intercellular adhesion molecule-3 (ICAM-3), leukointegrinadhesin, the platelet glycoprotein gp IIb/IIIa, cardiac myosin heavychain, parathyroid hormone, rNAPc2 (which is an inhibitor of factorVIIa-tissue factor), MHC I, carcinoembryonic antigen (CEA),alpha-fetoprotein (AFP), tumor necrosis factor (TNF), CTLA-4 (which is acytotoxic T lymphocyte-associated antigen), Fc-γ-1 receptor, HLA-DR 10beta, HLA-DR antigen, L-selectin, IFN-γ, Respiratory Syncitial Virus,human immunodeficiency virus (HIV), hepatitis B virus (HBV),Streptococcus mutans, and Staphylococcus aureus.

The formulations of the invention can also be used for anti-idiotypicantibodies, or substantially similar polypeptides, including but notlimited to anti-idiotypic antibodies against: an antibody targeted tothe tumor antigen gp72; an antibody against the ganglioside GD3; or anantibody against the ganglioside GD2.

The Fc domain containing polypeptide suitable for storage in the presentpharmaceutical composition can be produced by living host cells thatexpress the polypeptide, such as hybridomas in the case of antibodies,or host cells that that have been genetically engineered to produce thepolypeptide in the case of fusion polypeptides or antibodies. Methods ofgenetically engineering cells to produce polypeptides are well known inthe art. See, e.g., Ausubel et al., eds. (1990), Current Protocols inMolecular Biology (Wiley, New York). Such methods include introducingnucleic acids that encode and allow expression of the polypeptide intoliving host cells. These host cells can be bacterial cells, fungalcells, or, preferably, animal cells grown in culture. Bacterial hostcells include, but are not limited to, Escherichia coli cells. Examplesof suitable E. coli strains include: HB101, DH5α, GM2929, JM109, KW251,NM538, NM539, and any E. coli strain that fails to cleave foreign DNA.Fungal host cells that can be used include, but are not limited to,Saccharomyces cerevisiae, Pichia pastoris and Aspergillus cells. A fewexamples of animal cell lines that can be used are CHO, VERO, BHK, HeLa,Cos, MDCK, 293, 3T3, and WI38. New animal cell lines can be establishedusing methods well know by those skilled in the art (e.g., bytransformation, viral infection, and/or selection). Optionally, thepolypeptide can be secreted by the host cells into the medium.

Purification of the expressed Fc domain containing polypeptide can beperformed by any standard method. When the Fc domain containingpolypeptide is produced intracellularly, the particulate debris isremoved, for example, by centrifugation or ultrafiltration. When thepolypeptide is secreted into the medium, supernatants from suchexpression systems can be first concentrated using standard polypeptideconcentration filters. Protease inhibitors can also be added to inhibitproteolysis and antibiotics can be included to prevent the growth ofmicroorganisms.

The Fc domain containing polypeptide can be purified using, for example,hydroxyapatite chromatography, gel electrophoresis, dialysis, andaffinity chromatography, and any combination of purification techniquesknown or yet to discovered. For example, protein A can be used to purifyFc domain containing polypeptides that are based on human gamma 1, gamma2, or gamma 4 heavy chains (Lindmark et al., 1983, J. Immunol. Meth.62:1-13). Protein G is recommended for all mouse isotypes and for humangamma 3 (Guss et al., 1986, EMBO J. 5:1567-1575).

Other techniques for polypeptide purification such as fractionation onan ion-exchange column, ethanol precipitation, reverse phase HPLC,chromatography on silica, chromatography on heparin SEPHAROSET™,chromatography on an anion or cation exchange resin (such as apolyaspartic acid column), chromatofocusing, SDS-PAGE, and ammoniumsulfate precipitation can also be utilized depending on need.

Pharmaceutical Composition

The present pharmaceutical composition is prepared by combining, inaddition to a purified polypeptide described above, an aggregationinhibitor. Further, a buffer, a tonicity modifier and an additionalexcipient can be added as needed. It will be understood one of ordinaryskill in the art that the combining of the various components to beincluded in the composition can be done in any appropriate order,namely, the buffer can be added first, middle or last and the tonicitymodifier can also be added first, middle or last. It is also to beunderstood by one of ordinary skill in the art that some of thesechemicals can be incompatible in certain combinations, and accordingly,are easily substituted with different chemicals that have similarproperties but are compatible in the relevant mixture.

Aggregation inhibitors reduce a polypeptide's tendency to associate ininappropriate or unwanted ternary or quaternary complexes. Unexpectedly,the present inventors have found that the amino acids L-arginine and/or,L-cysteine, act to reduce aggregation of Fc domain containingpolypeptide in a formulation for long periods, e.g., two years or more.The concentration of the aggregation inhibitor in the formulation ispreferably between about 1 mM to 1M, more preferably about 10 mM toabout 200 mM, more preferably about 10 mM to about 100 mM, even morepreferably about 15 mM to about 75 mM, and yet more preferably about 25mM. These compounds are available from commercial suppliers.

Buffering agents maintain pH in a desired range and various bufferssuitable for use in the pharmaceutical composition of the inventioninclude histidine, potassium phosphate, sodium or potassium citrate,maleic acid, ammonium acetate, tris-(hydroxymethyl)-aminomethane (tris),various forms of acetate and diethanolamine. One preferred buffer issodium phosphate as its buffering capacity is at or near pH 6.2. Theconcentration of the buffer in the formulation is preferably betweenabout 1 mM to about 1M, more preferably about 10 mM to about 200 mM.Buffers are well known in the art and are manufactured by known methodsand available from commercial suppliers.

When the pH of the pharmaceutical composition is set at or nearphysiological levels comfort of the patient upon administration ismaximized. In particular, it is preferred that the pH be within a rangeof pH about 5.8 to 8.4, with about 6.2 to 7.4 being preferred, however,it is to be understood that the pH can be adjusted as necessary tomaximize stability and solubility of the polypeptide in a particularformulation and as such, a pH outside of physiological ranges, yettolerable to the patient, is within the scope of the invention.

A tonicity modifier is understood to be a molecule that contributes tothe osmolality of a solution. The osmolality of a pharmaceuticalcomposition is preferably regulated in order to maximize the activeingredient's stability and also to minimize discomfort to the patientupon administration. Where serum is approximately 300+/−50 milliosmolalsper kilogram. It is generally preferred that a pharmaceuticalcomposition be isotonic with serum, i.e., having the same or similarosmolality, which is achieved by addition of a tonicity modifier, thusit is contemplated that the osmolality will be from about 180 to about420 milliosmolals, however, it is to be understood that the osmolalitycan be either higher or lower as specific conditions require. Examplesof tonicity modifiers suitable for modifying osmolality include, but arenot limited to amino acids (e.g., arginine, cysteine, histidine andglycine), salts (e.g., sodium chloride, potassium chloride and sodiumcitrate) and/or saccharides (e.g., sucrose, glucose and mannitol). Theconcentration of the tonicity modifier in the formulation is preferablybetween about 1 mM to 1M, more preferably about 10 mM to about 200 mM.Tonicity modifiers are well known in the art and are manufactured byknown methods and available from commercial suppliers.

Excipients, also referred to as chemical additives, co-solutes, orco-solvents, that stabilize the polypeptide while in solution (also indried or frozen forms) can also be added to a pharmaceuticalcomposition. Examples include but are not limited to sugars/polyols suchas: sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose,inositol, trehalose, glucose; polymers such as: serum albumin (bovineserum albumin (BSA), human SA or recombinant HA), dextran, PVA,hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin,polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC); non-aqueoussolvents such as: polyhydric alcohols, (e.g., PEG, ethylene glycol andglycerol) dimethysulfoxide (DMSO) and dimethylformamide (DMF); aminoacids such as: proline, L-serine, sodium glutamic acid, alanine,glycine, lysine hydrochloride, sarcosine and gamma-aminobutyric acid;surfactants such as: Tween-80™ (polysorbate 80), Tween-20™ (polysorbate20), SDS, polysorbate, polyoxyethylene copolymer; and miscellaneousexcipients such as: potassium phosphate, sodium acetate, ammoniumsulfate, magnesium sulfate, sodium sulfate, trimethylamine N-oxide,betaine, metal ions (e.g., zinc, copper, calcium, manganese, andmagnesium), CHAPS, monolaurate, 2-O-beta-mannoglycerate or anycombination of the above.

The concentration of one or more excipients in a formulation of theinvention is/are preferably between about 0.001 to 5 weight percent,more preferably about 0.1 to 2 weight percent. Excipients are well knownin the art and are manufactured by known methods and available fromcommercial suppliers.

In one illustrative embodiment, a formulation of the invention cancomprise about 25 to about 50 mg TNFR:Fc (etanercept), about 10 mM toabout 100 mM L-arginine, about 10 mM to about 50 mM sodium phosphate,about 0.75% to about 1.25% sucrose, about 50 mM to about 150 mM NaCl, atabout pH 6.0 to about pH 7.0. In another embodiment L-arginine can bereplaced with L-cysteine (at about 1 to about 500 micromolar) in theformulation. In yet another embodiment, the pH can be about pH 7.0. Inanother specific embodiment, a formulation of the invention can compriseabout 25 mg/ml TNFR:Fc, about 25 mM L-arginine, about 25 mM sodiumphosphate, about 98 mM sodium chloride, and about 1% sucrose at about pH6.2.

In another embodiment, a formulation of the invention can comprise about10 to about 100 mg/mL of RANK:Fc in about 10 mM to about 100 mML-arginine, about 10 mM to about 50 mM sodium phosphate, about 0.75% toabout 1.25% sucrose, about 50 mM to about 150 mM NaCl, at about pH 6 toabout pH 7. In a specific embodiment, the formulation of the inventioncomprises 50 mg/ml RANK:Fc in about 25 mM L-arginine, about 25 mM sodiumphosphate, about 98 mM sodium chloride, and about 1% sucrose at about pH6.2.

In yet another embodiment, a formulation of the invention can comprisean effective amount of an Fc domain containing polypeptide, about 10 mMto about 100 mM L-arginine, about 10 mM to about 50 mM sodium phosphate,about 0 to 5% Mannitol and 0 to 0.2% Tween-20™ (polysorbate 20) at aboutpH 6 to 7. In another embodiment, a formulation of the invention cancomprise an effective amount of an antibody, such as Emab (an anti-CD22specific antibody), about 25 mM L-arginine, about 25 mM sodiumphosphate, about 4% Mannitol, about 0.02% Tween-20™ (polysorbate 20) andat about pH 6.0.

In yet another embodiment, the invention provides a method of treating amammal comprising administering a therapeutically effective amount ofthe pharmaceutical composition described herein, wherein the mammal hasa disease or disorder that can be beneficially treated with a Fc domaincontaining polypeptide in the composition. In yet another embodiment,the Fc domain containing polypeptide is derived from the same species ofmammal as is to be treated with the composition. In a particularembodiment, the mammal is a human patient in need of treatment. When theFc domain containing polypeptide of the composition is TNFR:Fc, examplesof diseases or disorders that can be treated include but are not limitedto rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis,Wegener's disease (granulomatosis), Crohn's disease (or inflammatorybowel disease), chronic obstructive pulmonary disease (COPD), HepatitisC, endometriosis, asthma, cachexia, psoriasis, and atopic dermatitis.Additional diseases or disorders that can be treated with TNFR:Fcinclude those described in WO 00/62790, WO 01/62272 and U.S. PatentApplication No. 2001/0021380, the relevant portions of which areincorporated herein by reference.

In yet another embodiment, the invention provides a method foraccelerated stability testing of the stability an Fc domain containingpolypeptide in a pharmaceutical composition of the invention comprisingthe steps of testing the activity of the polypeptide formulatedaccording to the invention prior to storage, i.e., time zero, storingthe composition at 37° C. for one month and measuring the stability ofthe polypeptide, and comparing the stability form time zero to the onemonth time point. This information is helpful for early elimination ofbatches or lots that appear to have good stability initially, yet do notstore well for longer periods.

Moreover, the present pharmaceutical composition provides improvedlong-term storage such that the active ingredient, e.g., an Fc domaincontaining polypeptide, is stable over the course of storage either inliquid or frozen states. As used herein, the phrase “long-term” storageis understood to mean that the pharmaceutical composition can be storedfor three months or more, for six months or more, and preferably for oneyear or more. Long term storage is also understood to mean that thepharmaceutical composition is stored either as a liquid at 2-8° C. or isfrozen, e.g., at −20° C. or colder. It is also contemplated that thecomposition can be frozen and thawed more than once. The term “stable”with respect to long-term storage is understood to mean that the activepolypeptide of the pharmaceutical composition does not lose more than20%, or more preferably 15%, or even more preferably 10%, and mostpreferably 5% of its activity relative to activity of the composition atthe beginning of storage.

Effective Dose of the Pharmaceutical Composition

The appropriate dosage, or therapeutically effective amount, of the Fcdomain containing polypeptide of the formulation will depend on thecondition to be treated, the severity of the condition, prior therapy,and the patient's clinical history and response to the therapeuticagent. The proper dose can be adjusted according to the judgment of theattending physician such that it can be administered to the patient onetime or over a series of administrations. The pharmaceutical compositioncan be administered as a sole therapeutic or in combination withadditional therapies as needed.

In one embodiment, the effective Fc domain containing polypeptide amountper adult dose ranges from about 1-500 mg/m², or from about 1-200 mg/m²,or from about 1-40 mg/m² or about 5-25 mg/m². Alternatively, a flat dosemay be administered, whose amount may range from 2-500 mg/dose, 2-100mg/dose or from about 10-80 mg/dose. If the dose is to be administeredmore than one time per week, an exemplary dose range is the same as theforegoing described dose ranges or lower and preferably administered twoor more times per week at a per dose range of 25-100 mg/dose. In anotherembodiment, an acceptable dose for administration by injection contains80-100 mg/dose, or alternatively, containing 80 mg per dose. The dosecan be administered at biweekly, weekly doses, or separated by severalweeks (for example 2 to 8). In this example TNFR:Fc (etanercept) isgenerally administered at 25 mg by a single subcutaneous (SC) injection.

In many instances, an improvement in a patient's condition will beobtained by a dose of up to about 100 mg of the pharmaceuticalcomposition one to three times per week over a period of at least threeweeks, though treatment for longer periods may be necessary to inducethe desired degree of improvement. For incurable chronic conditions theregimen may be continued indefinitely. For pediatric patients (ages4-17), a suitable regimen involves a dose of 0.4 mg/kg to 5 mg/kg of athe polypeptides of the invention, administered one or more times perweek.

In another embodiment, it is contemplated that the pharmaceuticalformulation of the invention is prepared in a bulk formulation and assuch, the components of the pharmaceutical composition are adjusted sothat it is higher than would be required for administration and dilutedappropriately prior to administration.

Administration of the Pharmaceutical Composition

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, i.e., subcutaneously,intramuscularly, intravenously, intraperitoneal, intracerebrospinal,intra-articular, intrasynovial, and/or intrathecal. Parenteraladministration can be by bolus injection or continuous infusion.Pharmaceutical compositions for injection may be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. In addition, a number of recent drug deliveryapproaches have been developed and the pharmaceutical compositions ofthe present invention are suitable for administration using these newmethods, e.g., Inject-ease™, Genject™, injector pens such as GenPen™,and needleless devices such as MediJector™ and BioJector™. The presentpharmaceutical composition can also be adapted for yet to be discoveredadministration methods. See also Langer, 1990, Science, 249:1527-1533.

The pharmaceutical composition can also be formulated as a depotpreparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the formulations may bemodified with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions may, if desired, be presented in a vial,pack or dispenser device which may contain one or more unit dosage formscontaining the active ingredient. In one embodiment the dispenser devicecan comprise a syringe having a single dose of the liquid formulationready for injection. The syringe can be accompanied by instructions foradministration.

In another embodiment, the present invention is directed to a kit orcontainer, which contains an aqueous pharmaceutical composition of theinvention. The concentration of the polypeptide in the aqueouspharmaceutical composition can vary over a wide range, but is generallywithin the range of from about 0.05 to about 20,000 micrograms permilliliter (μg/ml) of aqueous formulation. The kit can also beaccompanied by instructions for use.

The invention will be more fully understood by reference to thefollowing examples. The examples should not, however, be construed aslimiting the scope of the invention.

EXAMPLES Example 1

In order to determine the best excipient to prevent aggregation of an Fcdomain containing polypeptide, TNFR:Fc was produced and tested for lightscattering of a sample (Is) containing the TNFR:Fc with variousexcipients after incubation at 51° C.+/−1° C., and compared to lightscattering of a control (Ic) sample with TNFR:Fc alone stored at 2-8° C.The ratio is measured as Is/Ic, and a ratio of one represents atheoretical baseline where there is no change in the light scattering,i.e., aggregation, of the test compound. The various excipients testedincluded 5% ascorbic acid, 5% mannitol, 10% sucrose, 1%polyvinylpyrrolidone (PVP-K15), 0.1% polyethylene glycol (PEG, Mw=1000),0.6% ethanol, 1.2% glycine, 2% L-arginine, 0.01% Pluronic F68, 1.6%Betaine and 1.5% L-cysteine. Surprisingly, L-arginine was the onlyaggregation inhibitor found to keep the Is/Ic ratio below one for theentire 200 hour test period.

Example 2

TNFR:Fc produced and denoted as lots A, B, C and D were evaluatedagainst TNFR:Fc produced by a different method and having higher initialaggregation (lot 1) for stability in a liquid formulation (25 mMphosphate, 25 mM L-arginine, 98 mM NaCl, 1% sucrose, at pH 6.2) insyringes or glass vials at −70° C., −20° C., 2-8° C., 30° C., and 37° C.Samples were analyzed by size exclusion chromatography (SEC), denaturedSEC (dSEC), hydrophobic interaction chromatography (HIC), sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and forbinding and bioactivity at various timepoints. The bioactivity can bemeasured by any number of assays including by SEC, dSEC, HIC, bindingactivity and bioactivity, as discussed below.

Size Exclusion Chromatography:

SEC was used to assess the level of high molecular weight (HMW) species(aggregate that formed) in the samples during storage. Low molecularweight (LMW) species are better resolved by dSEC and that data can befound in the next section. FIG. 1 shows the SEC data for the samplesstored at 2-8° C. and FIG. 2 shows the SEC data for samples stored underaccelerated conditions of 37° C.

Data was also collected for samples stored at 30° C. (data not shown)and the levels of HMW species were intermediate to those seen at 2-8° C.and 37° C. During storage for 1 year at 2-8° C., aggregate levelsremained stable, or increased less than 0.6% in the worst case for lotA. No significant increases in aggregate were seen during storage at2-8° C. Under accelerated conditions during storage at 37° C., aggregatelevels in lot 1 increased to 19% during 12 months, and to 14% and 12% inlot A and B, respectively. The slope of the lines were very similar,showing that the molecules aggregated at the same rate, and that thedifferences between lots A-D and lot 1 are due to the initial levels ofaggregate, higher in lot 1 than lots A-D. For lot B, there was nodifference between samples stored in a vial at −70° C., in a syringe at−70° C., in a syringe at −20° C., or in a syringe after thermaltreatment and storage at −20° C. (data not shown). All values werewithin 0.4% of the −70° C. vial control (and the time 0 value) after 12months of storage.

Denatured Size Exclusion Chromatography:

Denatured SEC (dSEC) quantitation of the low molecular weight (LMW)species is shown in FIG. 3 for samples stored at 2-8° C., and FIG. 4 forsamples stored at 37° C. Lots A-D and lot 1 were analyzed by dSEC afterstorage for up to 1 year at 2-8° C., but lots C and D were not analyzedpast 6 months of storage under the accelerated conditions of 37° C.During storage at 37° C., lot 1 and lots A, B, and C showed similarbreakdown, while lot D showed higher breakdown during heat stressingthan lot 1 and the other lots. The similarity in lots A and B and lot 1was also seen during storage at 30° C. (data not shown), with levels ofbreakdown intermediate to that seen at 2-8° C. and 37° C. For lot B,there was no difference between samples stored in a vial at −70° C., ina syringe at −70° C., in a syringe at −20° C., or in a syringe afterthermal treatment and storage at −20° C. (data not shown). All valuesafter 12 months of storage at −70° C. and −20° C. were within 0.7% ofeach other and the time 0 value.

During storage at 30° C. (data not shown), the % LMW by dSEC for lot Atracks well with lot B, although both lots show slightly higher levelsof breakdown than lot 1 at this temperature. Lot D shows higher levelsof LMW species at both 2-8° C. and 37° C. at all timepoints, includingtime 0. The breakdown products in lot D appear to be larger in size thanis typically seen by dSEC analysis of stressed TNFR:Fc samples. Thesedifferent species are seen after storage at both 2-8° C. and 37° C.

Hydrophobic Interaction Chromatography:

HIC was used to separate various TNFR:Fc-related species. Peak 1 (and anearlier eluting peak denoted as pre-peak 1) has been shown to consistmainly of low molecular weight species. Peak 2 includes the folded,intact dimer (active). Peak 3 includes aggregated material and lessactive dimers.

HIC peak 1 data are shown in FIG. 5 for samples stored at 2-8° C. andFIG. 6 for samples stored at 37° C. For all lots except lot D, levels ofLMW species remains relatively constant (within 1.2% over 12 months) forsamples stored at 2-8° C. If the average value for the −70° C. samplesis used in place of the time 0 value for lot A, the curves for all lotsexcept lot D align well. Lot D shows more peak 1 than the other lots,corroborating the high levels of LMW species seen by dSEC. After heatstressing the samples at 37° C. for up to 1 year, lot B and lot 1 showapproximately 30% HIC peak 1, whereas lot A shows approximately 45% HICpeak 1. Lot D showed 47% HIC peak 1 after only 6 months of stressing at37° C.

As noted above, HIC peak 2 represents the most desired, active species.FIGS. 7 and 8 show the % HIC peak 2 for samples stored at 2-8° C. and37° C., respectively. Although lot 1 starts out at a lower initial %peak 2, it retains the level of active species during storage for 12months at 2-8° C. Lots A, B, and C also retain active species during the12 months of refrigerated storage. Under accelerated conditions of 37°C., all lots tested lose HIC peak 2 during storage.

HIC peak 3 levels remained essentially constant during 1 year of storageat 2-8° C. (FIG. 9). Variation in % peak 3 for all lots ranged between 1and 3%, well within the error of integration. For lots A, B, C and D,HIC peak 3 does not show baseline resolution, introducing morevariability in integration. For lot 1, the peak is more clearly defined.After storage at 37° C., the HIC peak 3 levels in lot 1 are morevariable, but remain fairly constant, except for a possible increase at12 months (FIG. 11). Between lots A-D, no clear differences were seenafter storage at 37° C., except for at 12 months, where lot B shows anincrease in HIC peak 3 level.

Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis:

SDS-PAGE analysis of samples stored for 12 months at −70° C., −20° C.,2-8° C., 30° C., and 37° C. was performed. Lot A had an increase inbands associated with both a ˜50 kD and ˜34 kD breakdown fragment afterstorage at 2-8° C. for 1 year. At elevated temperatures, extensivedegradation was seen, with many small molecular weight bands showingincreased intensities.

Lot 1 showed no change after 1 year of storage at 2-8° C., but showedincreased ˜50 kD and ˜34 kD breakdown fragment after 1 year at 30 and37° C. Lot B showed no changes during storage for 12 months at −70° C.(vial or syringe) or in syringes at −20° C., with or without thermaltreatment to eliminate supercooling. After 12 months at 2-8° C.,however, bands corresponding to both the ˜50 kD and ˜34 kD breakdownfragment fragments showed increased intensity. Storage at 30 or 37° C.for 1 year resulted in breakdown, with many small molecular weight bandsin addition to the previously discussed ˜50 kD and ˜34 kD breakdownfragment.

Lots C and D were analyzed after storage for 12 months at −70° C. and2-8° C. Lot 1, and lots B, C and D are were analyzed after storage for12 months at −70° C. and 2-8° C. and showed similar patterns ofdegradation as noted above.

Binding and Bioactivity:

FIG. 11 shows the binding activity data derived from an ELISA, for lotsA-D and lot 1 stored for 6 and 12 months at −70° C., 2-8° C., 30° C.,and 37° C. The error bars on the −70° C. samples indicate +/−30%. Onlyvalues outside of these error bars will be considered significant due toassay variability. Lots A and B retained full binding activity after 6months at 2-8 and 30° C., but at 12 months, only the samples stored at2-8° C. maintained full binding activity. Lot 1 was able to maintainfull activity for up to 12 months after storage at both 2-8 and 30° C.,despite showing LMW levels of 13.6% (by dSEC; data not shown) and 8% HMW(by SEC; data not shown) after 1 year at 30° C. Lots C and D alsoretained full binding activity after 1 year of storage at 2-8° C.,despite higher levels of breakdown products seen in lot D by dSEC andHIC.

An example of a TNFR:Fc bioassay is to inhibit the negative growthresponse of a cell line to human TNF-alpha. The presence of TNF-alphainhibits the cells from growing through induction of apoptosis. Thepresence of biologically active soluble rhuTNF receptor (TNFR:Fc)specifically neutralizes TNF-alpha in a dose-dependent manner. A TNFRreference standard, control, and samples are added and titrated in a 96well microtiter plate format. A known concentration of cells is added toeach well followed by addition of TNF-alpha. After an incubation period,non-adherent cells are removed by gently washing with phosphate bufferedsaline (PBS) and the remaining cells are stained. After an incubationperiod, each well is read. The units of each well are directlyproportional to the specific activity of TNFR. The results for thebioactivity assay (FIG. 12) corroborate the binding assay data.

CONCLUSIONS

Lots B and C formulated in a liquid phosphate formulation (25 mMphosphate, 25 mM L-arginine, 98 mM NaCl, 1% sucrose, pH 6.2) were shownto be as stable as lot 1 in the same formulation for 1 year at −70° C.or 2-8° C. Lots A-D showed less aggregation than lot 1, and wereequivalent in terms of breakdown into lower molecular weight species(less than 4% LMW by dSEC at 12 months). Both lot 1 and the lots A-Dshowed increased breakdown and aggregation at elevated temperatures of30 and 37° C., but the lots that performed equal to lot 1 for up to oneyear at 2-8° C. showed equivalence to lot 1 during heat stressing for 1year. Lot D was shown to be less stable in the accelerated assay withhigh levels of low molecular weight breakdown products.

The data from the accelerated stability testing at 30 and 37° C.corresponds with the long-term stability at 2-8° C., and hence providesa method to accelerate the testing of the long-term stability of aformulated polypeptide at low temperatures without requiring a long-termstability assessment. Samples of lot B stored frozen in syringes at −70°C. and −20° C. showed similar stability to samples stored frozen in avial at −70° C., supporting the use of an embodiment of a pre-filledsyringe stored frozen until delivery to the patient.

EQUIVALENTS AND REFERENCES

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

What is claimed is:
 1. A pharmaceutical composition that is a stableaqueous formulation comprising an Fc domain containing polypeptide andan aggregation inhibitor, wherein the aggregation inhibitor isL-arginine.
 2. The composition of claim 1, further comprising a buffer.3. The composition of claim 2, wherein the buffer is selected from thegroup consisting of sodium phosphate, histidine, potassium phosphate,sodium or potassium citrate, maleic acid, ammonium acetate,tris-(hydroxymethyl)-aminomethane (tris), acetate and diethanolamine. 4.The composition of claim 3, wherein the L-arginine is at a concentrationof from about 10 mM to about 100 mM.
 5. The composition of any one ofclaim 1, 2, 3 or 4, further comprising a tonicity modifier.
 6. Thecomposition of claim 5, wherein the tonicity modifier is selected fromthe group consisting of arginine, cysteine, histidine, glycine, sodiumchloride, potassium chloride, sodium citrate, sucrose, glucose andMannitol.
 7. The composition of claim 6, wherein the tonicity modifieris sodium chloride.
 8. The composition of any one of claim 1, 2, 3, or4, further comprising an excipient.
 9. The composition of claim 6,further comprising an excipient.
 10. The composition of claim 7, furthercomprising an excipient.
 11. The composition of claim 8, wherein theexcipient is selected from the group consisting of sucrose, lactose,glycerol, xylitol, sorbitol, Mannitol, maltose, inositol, trehalose,glucose, bovine serum albumin (BSA), human SA or recombinant HA,dextran, PVA, hydroxypropyl methylcellulose (HPMC), polyethyleneimine,gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC),polyethylene glycol, ethylene glycol, glycerol, dimethysulfoxide (DMSO),dimethylformamide (DMF), proline, L-serine, sodium glutamic acid,alanine, glycine, lysine hydrochloride, sarcosine, gamma-aminobutyricacid, Tween-20, Tween-80, SDS, polysorbate, polyoxyethylene copolymer,potassium phosphate, sodium acetate, ammonium sulfate, magnesiumsulfate, sodium sulfate, trimethylamine N-oxide, betaine, zinc ions,copper ions, calcium ions, manganese ions, magnesium ions, CHAPS,sucrose monolaurate, and 2-O-beta-mannoglycerate.
 12. The composition ofclaim 11, wherein the excipient is sucrose.
 13. A stable pharmaceuticalcomposition comprising about 10 mg/ml to about 100 mg/ml TNFR:Fc,L-arginine, sodium phosphate, sodium chloride and sucrose.
 14. Thecomposition of claim 13, wherein L-arginine is about 10 mM to about 75mM.
 15. The composition of claim 13, wherein sodium phosphate is about 5mM to about 100 mM.
 16. The composition of claim 13, wherein sodiumchloride is about 5 mM to about 200 mM.
 17. The composition of claim 13,wherein sucrose is about 0.5% to about 1.5%.
 18. The composition ofclaim 13, wherein pH is about 5.5 to about 7.8.
 19. The composition ofclaim 13, having 25 mg/ml TNFR:Fc, 25 mM L-arginine, 25 mM sodiumphosphate, 98 mM sodium chloride, 1% sucrose at pH 6.2.
 20. Thecomposition of any one of claim 1, 13 or 19, wherein the composition isliquid.
 21. The composition of claim 20, wherein the composition isfrozen.
 22. A method of formulating a composition comprising combiningisolated TNFR:Fc with L-arginine.
 23. The method of claim 22, furthercomprising the steps of combining a buffer, a tonicity modifier, and anexcipient with the composition.
 24. The method of claim 22 or 23,wherein the composition is liquid.
 25. A kit comprising a compositioncomprising an Fc domain containing polypeptide and L-arginine, andinstructions for use of said composition.
 26. The kit of claim 25,wherein the composition is liquid.
 27. The kit of claim 25 or 26,wherein the composition is stored in a pre-filled sterile syringe. 28.The kit of claim 27, wherein the syringe is stored at about −20° C. toabout −70° C.
 29. A method of treating a mammal in need thereofcomprising administering a therapeutically effective amount of thepharmaceutical composition of any one of claim 4 or
 13. 30. A method ofaccelerated stability testing of an Fc domain containing polypeptide ina pharmaceutical composition, wherein the composition comprisesL-arginine, the steps of the method comprising storing the compositionat 37° C. and measuring the stability of the polypeptide after at leastone month at 37° C.